Discovery of restriction enzymes made gene cloning possible using recombinant DNA techniques. Genetic engineering has lead to the remarkable expansion of various industries and studies. Numerous restriction enzymes that can selectively cleave specific nucleotide sequences are known. These enzymes, essential tools for genetic engineering, are widely used for analysis of gene structures and the like.
For restriction enzymes, only naturally occurring restriction enzymes have been conventionally used, and gene recombination techniques using the naturally occurring restriction enzymes are limited to those targeting small DNAs such as plasmids. However, many DNAs are far larger than plasmids, and treating large DNA using conventionally-available restriction enzymes results in fragmentation due to cleavage at a considerably large number of sites, making desired gene recombination procedures impracticable. For this reason, it has been desired to provide a means for freely manipulating large DNA. In particular, it has been desired to provide a restriction enzyme that can selectively cleave a specific nucleotide sequence in a large DNA molecule.
For example, Japanese Patent Unexamined Publication (Kokai) No. 2005-143484 discloses a method of allowing two peptide nucleic acids (PNAs) to invade into DNA to activate a phosphodiester bond at a desired site in the DNA and then adding a Ce(IV)-EDTA complex to cleave the nucleic acid in an active site (hot spot)-selective manner. In the above method, sequences and lengths of the PNAs used for the formation of the hot spot are not limited, and therefore, it is considered that DNA of any size can be precisely cleaved at a desired site. However, no practical example of desired intracellular cleavage was demonstrated.
Fusion proteins are known in which an enzyme, or enzyme active site that has a nucleic acid cleaving activity, is bound to a DNA-binding protein such as a zinc finger protein (ZEP). For example, a fusion protein in which a zinc finger protein is bound to a catalytically-active site of the restriction enzyme FokI was presented by Kim et al. (Kim, Y., et al., Proc. Natl. Acad. Sci. USA, 93, pp. 1156-1160, 1996). Bibikova et al. reported homologous recombination in cells of frog, Drosophila and human using a chimeric zinc finger nuclease (ZFN) (Bibikova M., et al., Science, 300, p. 764, 2003). For ZFN, it can be directed to cleave different target sites by replacing a zinc finger domain with another domain having different specificity. Ideally, this nuclease can therefore be designed to target any arbitrary sequence on a genome (Nature Methods 2, 405 (2005), doi: 10.1038/nmeth0605-405).
However, to cleave a nucleic acid using the aforementioned ZFN, two ZFN molecules need to act in a cooperative manner. That is, the two ZFN molecules bind oppositely to a desired site on a DNA and a double-stranded DNA is nicked, and then homologous recombination occurs between a chromosome and a donor DNA molecule to cause replacement with a desired nucleotide sequence. Therefore, for example, when a long region of a DNA chain is removed for gene therapy or the like, a problem arises that a set of two ZFN molecules is needed for each of two cleavage sites to perform homologous recombination. The ZFN molecule has a structure in which an enzyme cleavage site is bound to an end of a zinc finger protein (including a zinc finger protein having three, four, or six zinc finger domains). This molecule will not reduce affinity for a target site after the cleavage of a nucleic acid and remains bound to the nucleic acid. Therefore, the molecule fails to catalytically exert the cleavage action by repeating a cycle of binding and successive cleavage, followed by dissociation and rebinding.